PEDF基因修饰人脐带间充质干细胞对糖尿病大鼠视网膜的保护作用

The protective effect of pigment epithelial-derived factor modified human umbilical cord mesenchymal stem cells on rats with diabetic retinopathy

目的 探讨色素上皮衍生因子（PEDF）基因修饰的人脐带间充质干细胞（hUCMSC）对糖尿病视网膜病变（DR）大鼠视网膜的保护作用.方法 实验研究.采用慢病毒包装的PEDF-MSC-绿色荧光蛋白（GFP）质粒和GFP-间充质干细胞（MSC）质粒转染hUCMSC,并测量PEDF及VEGF的表达.成年SD大鼠50只采用随机数字表法随机分为5个组：正常对照组（A组）、DR对照组（B组）、磷酸盐缓冲液（PBS）治疗组（C组）、GFP-MSC治疗组（D组）及PEDF-MSC治疗组（E组）,各组均为10只大鼠.选取链脲佐菌素（STZ）腹腔注射诱导糖尿病大鼠模型,造模成功后4个月进行干预治疗,D组与E组分别给予玻璃体腔注射GFP-MSC和PEDF-MSC,C组给予玻璃体腔注射PBS,A组与B组不予特殊治疗.干预后8周行HE染色观察视网膜内丛状层、内核层及外核层,并进行厚度测量,免疫组织化学染色观察视网膜PEDF和VEGF阳性染色情况,实时定量聚合酶链反应（RT-PCR）检测大鼠视网膜PEDF和VEGF mRNA的表达变化.计量数据均符合正态分布.两组数据间比较采用独立样本t检验;组间的数据采用单因素方差分析,组间多重比较采用LSD-t检验,两个变量之间相关性分析采用Pearson相关性检验.结果 胰蛋白酶消化法获取的hUCMSC表达CD105、CD73和CD90,不表达CD34、CD45、CD11b、CD19和HLA-DR.ELISA检测结果显示,PEDF-MSC-GFP的PEDF（84.09±7.07） μg/L较GFP-MSC （9.03±0.14） μg/L表达增高,差异均有统计学意义（P〈0.05）,而VEGF表达差异无统计学意义（P〉0.05）.视网膜厚度测量分析结果显示：玻璃体腔注射后2个月,E组内丛状层厚度显著下降,内核层与外核层厚度显著增加（P〈0.05）.玻璃体腔注射后2周,荧光显微镜下观察到D组与E组大鼠玻璃体腔内放射状及簇状排列的绿色荧光,视网膜中未发现明显绿色荧光.免疫组织化学法染色结果显示：治疗后2个月,E组PEDF平均A值增加;VEGF平均A值下降（P〈0.05）.RT-PCR结果显示：治疗后2个月,E组PEDF mRNA表达水平增加.VEGF mRNA表达水平下降（P〈0.05）.结论 玻璃体腔注射PEDF-MSC能上调早期糖尿病大鼠视网膜PEDF表达与下调VEGF表达,改善DR大鼠神经视网膜损伤.

Objective To investigate the effect of pigment epithelial-derived factor （PEDF） gene-modified human umbilical cord mesenchymal stem cells （MSC） on rats with diabetic retinopathy （DR）.Methods Experimental study.Human umbilical cord MSC were transfected by lentivirus packaging PEDF-MSC-green fluorescent protein （GFP） and GFP-MSC plasmid vectors,and the expression of PEDF and vascular endothelial growth factor （VEGF） was measured in the cell culture medium.Fifty adult male Sprague-Dawley rats were randomly divided into five groups：normal control group （group A）,DR control group （group B）,phosphate-buffered saline （PBS） treated group （group C）,GFP-MSC treated group （group D） and PEDF-MSC-GFP treated group （group E）,with 10 rats in each group.Streptozotocin was intraperitoneally injected to make early DR models.After four-month intervention,groups D and E were given intravitreal injection of GFP-MSC and PEDF-MSC-GFP;group C was given intravitreal injection of phosphate-buffered saline;groups A and B did not receive special treatment.The changes of retina in different groups were detected by hematoxylin and eosin staining,and the thickness of inner plexiform layer,inner nuclear layer and outer nuclear layer was measured by computer-based image analytical system.Immunohistochemistry was applied to observe PEDF and VEGF.Real-time quantitative polymerase chain reaction was used to detect the expression of PEDF and VEGF mRNA.Results The expression of CD105,CD73 and CD90 was positive,while the expression of CD34,CD45,CD11b,CD19 and HLA-DR was negative.ELISA results showed that after transfection PEDF protein expression in the supernatant of PEDF-MSC （84.09±7.07）tg/L was higher than the control group （9.03±0.14）μg/L （P〈0.05）.At 2 weeks after intravitreal injection,green fluorescence was observed in the rat vitreous of groups D and E under a fluorescence microscope;no obvious green fluorescence was found in the retina.After 2 months of intravitreal injection,the thickness of inner plexiform layer in group E was significantly decreased;the thickness of inner nuclear layer and outer nuclear layer was higher （P〈0.05）.Immunohistochemical staining showed that 2 months after intravitreal treatment,the average optical density values of PEDF were improved,but the average optical density values of VEGF were decreased in group E （P〈0.05）.Real-time polymerase chain reaction showed that 2 months after treatment,the expression level of PEDF mRNA in group E was improved,but the expression level of VEGF mRNA was decreased （P〈0.05）.Conclusions Intravitreal injection of PEDF-MSC could up-regulate the expression of PEDF and down-regulate the expression of VEGF in diabetic rats and may represent a novel candidate resource for cell therapy of DR nerve damage.